ALGR2 IS AN ATP GTP-DEPENDENT PROTEIN-KINASE INVOLVED IN ALGINATE SYNTHESIS BY PSEUDOMONAS-AERUGINOSA/

The exopolysaccharide alginate is a major virulence factor in the path ogenicity of Pseudomonas aeruginosa infecting the lungs of cystic fibr osis (CF) patients. Alginate synthesis by P. aeruginosa is believed to occur in response to environmental signals present in the CF lung. Tr anscription of a critical alginate biosynthetic gene, algD, is trigger ed by environmental signals and is known to be controlled by regulator y proteins AlgR1, AlgR2, and AlgR3. AlgR1 is a member of the family of response regulators of the phosphorylation-dependent two-component ba cterial signal transduction systems. In this report, we describe the c haracterization of AlgR2 as the kinase involved in phosphorylation of AlgR1. AlgR2, an 18-kDa soluble protein undergoes rapid autophosphoryl ation in the presence of either ATP or GTP and transfers the phosphate to AlgR1. AlgR2 retains high affinity for both ATP and GTP with an ap parent K(m) of 137 and 249 nM, respectively, for phosphorylation by th ese two substrates. ADP and GDP exhibit competitive inhibition with an apparent K(i) of 94 and 314 nM, respectively, during phosphorylation by ATP and 481 and 273 nM during phosphorylation by GTP. AlgR1 and Alg R2 can be isolated in the form of an 80-kDa complex that is capable of undergoing phosphorylation and intracomplex phosphotransfer in vitro. A 16-kDa AlgR2 analog, capable of autophosphorylation in the presence of ATP or GTP and transferring the phosphate moiety to AlgR1, has bee n characterized in Escherichia coli.